Chitosan Imparts Better Biological Properties for Poly(-caprolactone) Electrospun Membranes than Dexamethasone

Adhesion and proliferation assay The ADSC cell response on the membrane surfaces was investigated after 1 and 7 days of culture in growth media. Cell adhesion and proliferation tests were evaluated by staining the cells with 5-chloromethyl fluorescein diacetate (CMFDA; Life Technologies), rhodamine phalloidin, and 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen) to visualize cytoplasm, cytoskeleton, and nucleus, respectively, using a fluorescence microscope (Zeiss). Before staining, unadhered cells were aspirated and the substrates were gently rinsed (twice) with phosphatebuffered saline (PBS) before being transferred to a new 48-well plate. The samples were incubated (37 °C in 5% CO2) with a 10 μmol L -1 solution of CMFDA in PBS for 45 min. Following incubation, the solution was aspirated and the substrates incubated in growth media at 37 °C under 5% CO2 for 30 min. The media was aspirated and the


Cell culture (before seeding)
Adipose-derived mesenchymal stem cells (ADSCs) are cultured at 5% CO 2 atmosphere in a 175 cm 2 surface area of polystyrene vented tissue-culture flasks at 37 °C. The cell growth media comprises Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were seeded onto samples at a concentration of 5,000 cells per well in 48-well plates and cultured at 37 °C in 5% CO 2 .

Adhesion and proliferation assay
The ADSC cell response on the membrane surfaces was investigated after 1 and 7 days of culture in growth media. Cell adhesion and proliferation tests were evaluated by staining the cells with 5-chloromethyl fluorescein diacetate (CMFDA; Life Technologies), rhodamine phalloidin, and 4',6-diamidino-2-phenylindole (DAPI; Invitrogen) to visualize cytoplasm, cytoskeleton, and nucleus, respectively, using a fluorescence microscope (Zeiss).
Before staining, unadhered cells were aspirated and the substrates were gently rinsed (twice) with phosphatebuffered saline (PBS) before being transferred to a new 48-well plate. The samples were incubated (37 °C in 5% CO 2 ) with a 10 μmol L -1 solution of CMFDA in PBS for 45 min. Following incubation, the solution was aspirated and the substrates incubated in growth media at 37 °C under 5% CO 2 for 30 min. The media was aspirated and the ____________________________________________________ *e-mail: sharise_beatriz@hotmail.com; afmartins@utfpr.edu.br substrates rinsed (once) with PBS before transport to a new 48-well plate, where the cells were fixed in an aqueous 3.7% v/v formaldehyde solution for 15 min (25 °C). The fixative was aspirated and the substrates rinsed (thrice in PBS for 5 min per rinse) before addition to a new 48-well plate. The cells were permeabilized in an aqueous 1.0% v/v Triton X solution for 3.0 min (25 °C). This media was aspirated and the substrates rinsed (thrice), transferred to a new 48-well plate and incubated in an aqueous 5.0 μL mL -1 rhodamine-phalloidin solution for 25 min at 25 °C.
Then, an aqueous 1.0 μL mL -1 DAPI solution was added to each 48-well plate and incubated for 5.0 min. The solution was aspirated, and the substrates rinsed with PBS (twice), stored in a PBS buffer and placed in a lightresistant container at 20 °C. Analysis of the fluorescence was performed with ImageJ software. Cell count numbers were statistically analyzed using Tukey's test at a 5% significance level.

Cell morphology
SEM was used to compare the attachment and morphology of cells on the scaffolds after cell culture assay.
After proliferation and adhesion assays (1 and 7 days), the adhered cells to the samples were fixed in an aqueous 3.0% v/v glutaraldehyde, 0.10 mol L -1 sodium cacodylate, and 0.10 mol L -1 sucrose solution for 45 min (25 °C).
Then, the samples were soaked for 10 min in a buffer solution based on 0.10 mol L -1 sodium cacodylate and 0.10 mol L -1 sucrose. Sample surfaces and cells were processed in sequential ethanol dehydration for 10 min each, following dehydration in hexamethyldisilazane (10 min) and stored in a desiccator until SEM imaging. SEM images were determined from the same conditions reported in the sub-section "Characterization".  Figure S1. Release curve of dexamethasone obtained in terms of fraction released, which is obtained from poly(εcaprolactone) loaded with dexamethasone. The release study is evaluated in PBS (pH 7.4) at 37 °C (n = 2).